Meet the Expert Evening Meeting, Nov 2012    (Jordan, Lebanon, Qatar, Bahrain, Kuwait)  Sandro Esteves, M.D., Ph.D.       ...
Learning Objectives           WHO has updated reference limits for semen          analysis results. Understand how to adop...
Esteves SC, 2012       Review this lecture at:http://www.androfert.com.br/review
Spermatogenesis          Where do we stand compared to our relatives?                     Chimpanzee   Human       Gorilla...
Semen Characteristics Values                 of 1,953 Recent Fathers                (whose partners had a TTP* ≤ 12 months...
Semen analysis:                  New WHO Standards      Semen Parameter                  WHO 1999 WHO 20101      Volume (m...
New WHO standards          Caution to Adopt and Interpret Results  7Esteves
Do they represent global semen characteristics of fertile men?                                                 Oslo       ...
New WHO standards                   Reasons for Lower Cut-off Values              Different way of generating data        ...
New WHO Standards: Implications                    • Do we need to recall previous semen                      analysis rep...
New WHO Standards: Interpretation                     Focus on the 5th percentile          Laboratories seeking          t...
New WHO Standards          Not accurate to discriminate fertile and                       infertile men                   ...
New Diagnostic Tests          • Beyond routine semen analysis               Sperm DNA Integrity               Reactive Oxy...
Sperm DNA Integrity                               Testing                           • Normal sperm chromatin essential for...
Sperm DNA Fragmentation and                Assisted Conception              Live Birth Rates by                Pregnancy b...
Sperm DNA Fragmentation and                    Miscarriage       • Population: Meta-analysis of 16 cohort studies (2,969 c...
Quick and Easy Test for Sperm          DNA Fragmentation Assessment          Sperm Chromatin     Sperm with intact DNA sho...
Y Chromosome Microdeletion                     Screening                                                         Genetic C...
Predictive Value of Yq           Microdeletion Screening for             Sperm Retrieval in NOA           AZFa            ...
Oral Antioxidant Therapy                               for Male Infertility             Outcome            N              ...
Rationale for Antioxidants   in Male Infertility:   Oxidative Stress       30%-80% of infertile men       have elevated ma...
Quick and Easy Test for                          Reactive Oxygen Species                                Assessment        ...
How to use Antioxidants                                     Treatment Strategy       Once OS is diagnosed,                ...
Oral Antioxidants in Male    Our formula                 Infertility           Vitamin C 500mg           Vitamin E 400 mg ...
Azoospermia                    • It is not a synonymous of sterility          • Complete absence of sperm in the ejaculate...
Obstructive Azoospermia                   • Microsurgical       Potentially reconstruction        treatable • TURED       ...
Sperm Retrieval and ICSI Outcome                                    Obstructive                                  Azoosperm...
Non-obstructive Azoospermia                                                              Sperm                • Sperm prod...
29      Available at http://androfert.com.br/videosEsteves
30Esteves   Adapted from: Verza Jr & Esteves. Atlas of Human Reproduction, SBRH 2012
Sperm Retrieval in Testicular Failure  Novel Sperm   Retrieval  Methods forIdentification of                              ...
Sperm Retrieval and Reproductive          Potential of Azoospermic Men in ART                               97.9%         ...
Key Messages (1)             WHO references for semen analysis                           have been lowered.           Exer...
Key Messages (2)                Oxidative stress is a risk factor for                  male infertility and miscarriage.  ...
Current Scenarios of Male Infertility for IVF Specialists
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Current Scenarios of Male Infertility for IVF Specialists

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Current Scenarios of Male Infertility for IVF Specialists

  1. 1. Meet the Expert Evening Meeting, Nov 2012 (Jordan, Lebanon, Qatar, Bahrain, Kuwait) Sandro Esteves, M.D., Ph.D. Director, ANDROFERTCenter for Male Reproduction and Infertility Campinas, BRAZIL
  2. 2. Learning Objectives WHO has updated reference limits for semen analysis results. Understand how to adopt and interpret the new standards. New diagnostic tests go far beyond routine semen analysis. Learn which tests are available and how to use them. Antioxidant supplementation is a hot issue in male infertility. Understand the rationale of its use and how it can benefit your patients. Learn what is available for azoospermic men seeking fertility. Biological fatherhood is 2 possible even in the worst scenarios.Esteves
  3. 3. Esteves SC, 2012 Review this lecture at:http://www.androfert.com.br/review
  4. 4. Spermatogenesis Where do we stand compared to our relatives? Chimpanzee Human Gorilla 45 Kg 80 Kg 270 Kg 64 cc 20 cc 14 cc >1 ? 5 billion/mL million/mL 4Esteves
  5. 5. Semen Characteristics Values of 1,953 Recent Fathers (whose partners had a TTP* ≤ 12 months) Centiles 5% 50%* 95% Volume (mL) 1.5 3.7 6.8 Sperm count per mL (x106) 15.0 73.0 213.0 Sperm count per ejaculate (x106) 39.0 255.0 802.0 % Motility (total) 40 61 78 % Motility (progressive) 32 55 72 % Normal Morphology (strict criteria) 4 15 44 % Alive (eosin-nigrosin staining) 58 79 91 *TTP: time to pregnancy 5 Cooper et al: World Health Organization reference values for human semen characteristics. Hum Reprod Update 16: 231-245, 2010Esteves
  6. 6. Semen analysis: New WHO Standards Semen Parameter WHO 1999 WHO 20101 Volume (mL) ≥ 2.0 1.5 Count (x106/mL) ≥ 20 15 Total sperm number per ejaculate ≥ 40 39 Motility (%) ≥ 50 (a + b) 32 (a+b) Vitality (%) ≥ 75 58 Morphology (%)2 (14) 4 Leukocytes (x106/mL) < 1.0 <1.0 1LowerLimit (5% percentile), Recent fathers; 2Strict criteria Grade a = rapid progressive motility; Grade b = slow/sluggish progressive motility 6Esteves
  7. 7. New WHO standards Caution to Adopt and Interpret Results 7Esteves
  8. 8. Do they represent global semen characteristics of fertile men? Oslo Turku Norway Finland Columbia, Minneapolis Edimburgh USA UK Copenhagen Paris Denmark France Melbourne Australia Critical Appraisal of the WHO New Reference Values for Human Semen and Impact on Diagnosis and Treatment of Subfertile Men 8 Esteves, Zini, Aziz et al, Urology, in pressEsteves
  9. 9. New WHO standards Reasons for Lower Cut-off Values Different way of generating data • Method for semen analysis (QC standards) • Adoption of strict criterion for morphology • Single specimen of each individual Population Studied • Recent fathers with known TTP • Selection bias 9 Esteves et al. Critical Appraisal of the WHO New Reference Values for Human Semen and Impact on Diagnosis and Treatment of Subfertile Men. Urology 2012; 79(1):16-22.Esteves
  10. 10. New WHO Standards: Implications • Do we need to recall previous semen analysis reports ? Couples (N=987) with infertility duration > 12 months Abnormal results WHO 1999 Reclassified as “Normal” Morphology by strict WHO 2010 criterion accounted for (38.7%) 53% of reclassification 10Esteves Source: ANDROFERT, Brazil
  11. 11. New WHO Standards: Interpretation Focus on the 5th percentile Laboratories seeking to adopt the new standard should determine the strategy that would aid the clear communication of the clinical significance of the results. 11 Esteves & Agarwal Impact of the new WHO guidelines on diagnosis and practice of male infertility. Open Reprod Sci J 2011; 3: 7-15.Esteves
  12. 12. New WHO Standards Not accurate to discriminate fertile and infertile men Male infertility workup goes far beyond a simple semen analysis. History taking, physical examination and laboratory and sperm function testing are minimum standards. Esteves, Miyaoka & Agarwal. An update on the initial assessment of the infertile male. 12 CLINICS 2011; 66:1-10.Esteves
  13. 13. New Diagnostic Tests • Beyond routine semen analysis Sperm DNA Integrity Reactive Oxygen Species Y Chromosome Microdeletion Screening 13 Esteves et al. What the gynecologist should know about male infertility: an update. Arch Gynecol Obstet 2012; 286(1): 217-29Esteves
  14. 14. Sperm DNA Integrity Testing • Normal sperm chromatin essential for Background paternal genetic transmission • Unexplained infertility Sperm DNA Fragmentation • Recurrent pregnancy loss • Poor outcomes in IUI and IVF • Quantification of sperm DNA strand Principle breaks Specimen • Semen, spermatozoa • Nuclear dyes (Acridine orange, SCSA) Techniques • Direct assessment (TUNEL, COMET) • Nuclear matrix assays (SCD) 14 Esteves SC & Agarwal A. Novel concepts in male infertility.Esteves Int Braz J Urol 2011; 37:5-15.
  15. 15. Sperm DNA Fragmentation and Assisted Conception Live Birth Rates by Pregnancy by Method in Cases Intrauterine Insemination of Elevated Sperm DNA Fragmentation 42% 19% P <0.05 26% OR = 0.07 [95% CI: 0.01-0.48] 1.5% Normal Elevated IVF ICSI 15 Adapted from Bungum et al., Hum Reprod 2007Esteves
  16. 16. Sperm DNA Fragmentation and Miscarriage • Population: Meta-analysis of 16 cohort studies (2,969 couples), 14 prospective. • Techniques for DNA integrity: Acridine orange-based assays, TUNEL and COMET. • Findings: Significant increase in miscarriage rates in patients undergoing IVF/ICSI with high sperm DNA damage compared with those with low DNA damage. Risk ratio (RR) = 2.16 (1.54, 3.03), P < 0.00001). Miscarriage rates are positively correlated with sperm DNA damage levels 16 Robinson L et al. Hum Reprod. 2012; 27(10): 2908-17Esteves
  17. 17. Quick and Easy Test for Sperm DNA Fragmentation Assessment Sperm Chromatin Sperm with intact DNA show “halos”: Dispersion (SCD) Represent dispersed chromatin without breaks Sperm with fragmented DNA do not show “halos” : Represent DNA strand breaks Semen/Spermatozoa Quantitative: Normal: <30% cells with fragmented DNA 17 Esteves et al. What the gynecologist should know about male infertility: an update. Arch Gynecol Obstet 2012; 286(1): 217-29Esteves
  18. 18. Y Chromosome Microdeletion Screening Genetic Causes of • Deletions linked to Male Infertility Background spermatogenic failure 5% • Severe oligozoospermia 10% Who? and NOA 5% • PCR of the long arm of Principle Y-chromosome 80% Specimen • Peripheral blood Klinefelter Syndrome (47,XXY) Clinical • Diagnosis and Y-chromosome microdeletion predictive value for Significance sperm retrieval Congenital Vas Absence 18 Esteves SC & Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.Esteves
  19. 19. Predictive Value of Yq Microdeletion Screening for Sperm Retrieval in NOA AZFa AZFb AZFc deletion deletion deletion No retrievable No retrievable Sperm retrieved in sperm sperm 70% of cases 19 Esteves SC & Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.Esteves
  20. 20. Oral Antioxidant Therapy for Male Infertility Outcome N N Effect size studies participants (OR; 95% CI) Live birth 3 214 4.85 [1.92, 12.24] Pregnancy rate 15 964 4.18 [2.65, 6.59] DNA fragmentation 1 64 -13.80 [-17.50, -10.10] Miscarriage, sperm 6-16 242-700 No effect count, sperm motility Adverse effects 6 426 No effect Improve the outcomes of live birth and pregnancy rate for subfertile couples undergoing ART cycles 20 Showell MG et al. Antioxidants for male subfertility.Esteves Cochrane Database Syst Rev 2011 Jan 19;(1):CD007411.
  21. 21. Rationale for Antioxidants in Male Infertility: Oxidative Stress 30%-80% of infertile men have elevated markers of OS Agarwal et al., Urology 2006 Seminal Reactive Oxygen Species (ROS) (Log ROS + 1; cpm) 2,5 2 1,5 1 0,5 0 Fertile Infertile 21 Pasqualotto et al., Fertil Steril 2000Esteves
  22. 22. Quick and Easy Test for Reactive Oxygen Species Assessment Detection of Superoxide Anion Principle: reduction of nitroblue tetrazolium into formazan (coloured intermediate) Semen or seminal plasma Qualitative colorimetric 22 Tunc et al. Development of the NBT assay as a marker of sperm oxidative stress.Esteves Int J Androl 2010; 33(1): 13-21.
  23. 23. How to use Antioxidants Treatment Strategy Once OS is diagnosed, Differentiate between focus on identifying Select antioxidant sperm and leukocyte and controlling source formulation and dosage source of ROS of increased ROS Varicocele Ascorbic acid (Vit. C) Testing for α- tocopherol (Vit. E) Genital Infection Glutathione Leukocytes in Semen N-acetyl-cysteine Smoking Carnitine Medication Coenzyme Q10 Drug abuse Lycopene Picnogenol Systemic diseases Pentoxifylline Pollution Selenium Shao-Fu-Zhu-Yu-Tang Radiation Astaxanthin Lepidium meyenii α -linolenic acid and lignans Folic acid 23 ZincEsteves
  24. 24. Oral Antioxidants in Male Our formula Infertility Vitamin C 500mg Vitamin E 400 mg Folic acid 2 mg Zinc 25 mg Selenium 26 mcg How long: minimum 2 months From initiation of sperm production to ejaculation Old concept ~80 days New concept ~60 days Misell LM et al. J Urol. 2006 24 Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011; 37 (1): 5-15Esteves
  25. 25. Azoospermia • It is not a synonymous of sterility • Complete absence of sperm in the ejaculate • 1-3% male population; ~10-15%% male infertility Non- Obstructive obstructive • Sperm production deficient or • Normal sperm production absent • Mechanical blockage • Cryptorchidism, Radiation, • Vasectomy, Post-infectious, Chemotherapy, Trauma, Congenital Genetic, Orchitis, Varicocele, Gonadotoxins, Unexplained 25Esteves
  26. 26. Obstructive Azoospermia • Microsurgical Potentially reconstruction treatable • TURED Sperm • Epididymis retrieval • Testis for ART • Simple and effective 26 Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the ICSI Era. CLINICS 2011; 66:1463-77.Esteves
  27. 27. Sperm Retrieval and ICSI Outcome Obstructive Azoospermia 265 277 250 CBAVD Post-vasectomy Post-infection 34.4 32.2 36.4 35.8 37.0 35.5 % Live birth Gestational age (wks) Birth weight (gramsx10) 27 Esteves et al. J Urol 12013; 189: http://dx.doi.org/10.1016/j.juro.2012.08.084Esteves
  28. 28. Non-obstructive Azoospermia Sperm • Sperm production Untreatable reduced or absent Retrieval condition • Geographic location for ART unpredictable TESE Micro-TESE Conventional TESA • 15%- • 20%- • 40%- 50% 63% 67% SRR SRR SRR 28 Friedler et al. 1997; Ezeh et al. 1998; Schlegel 1999; Amer et al. 2000; Okada et al. 2002; Okubu et al.Esteves 2002; Tsujimura et al. 2002; Ramon et al. 2003; Verza Jr. & Esteves 2011.
  29. 29. 29 Available at http://androfert.com.br/videosEsteves
  30. 30. 30Esteves Adapted from: Verza Jr & Esteves. Atlas of Human Reproduction, SBRH 2012
  31. 31. Sperm Retrieval in Testicular Failure Novel Sperm Retrieval Methods forIdentification of Confocal fluorescence microscopySpermatogenic (in vivo; murine model) Sites Multi-photon microscopy (ex vivo; human model) Full-field optical coherence tomography (ex vivo; rat model) Najari et al, J Urol 2012; Smith et al J Urol 2012; Ramasamy et al., J Pathol Inform 2012
  32. 32. Sperm Retrieval and Reproductive Potential of Azoospermic Men in ART 97.9% Obstructive (N=142) Non-obstructive (N=172) 55.2% 38.2% 25.0% Sperm Retrieval Live Birth Odds ratio 43.0 1.86 95% CI 10.3 – 179.5 1.03 – 2.89 P-value <0.01 0.03 Prudencio C, Seoul B, Esteves SC. Reproductive potential of azoospermic men undergoing intracytoplasmic sperm injection is dependent on the type of azoospermia. 32 Fertil Steril 2010; 94(4):S232-3.Esteves
  33. 33. Key Messages (1) WHO references for semen analysis have been lowered. Exercise caution to interpret results for they have important shortcomings. New diagnostic tests allow assessment to sperm DNA integrity, seminal ROS and Y-chromosome genetic infertility. Tests results aid in the clinical management and/or have prognostic value in ART. 33Esteves
  34. 34. Key Messages (2) Oxidative stress is a risk factor for male infertility and miscarriage. Antioxidant supplementation improve the chances for pregnancy and live birth for couples undergoing ART. Azoospermia is not synonymous of sterility. Treatment options include microsurgery and sperm retrieval coupled with ICSI. Reproductive potential in ART is 34 dependent on the type of azoospermia.Esteves

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